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Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

ABSTRACT: BACKGROUND: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. AIM: The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. RESULTS: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. CONCLUSIONS: Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.

SUBMITTER: Tanner GJ

PROVIDER: S-EPMC3585327 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

Tanner Gregory J GJ Blundell Malcolm J MJ Colgrave Michelle L ML Howitt Crispin A CA

PloS one 20130228 2

<h4>Background</h4>Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed.<h4>Aim</h4>The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.<h4>Results</h4>A simple alcohol-dithiothreitol extract ...[more]

PMID: 23509607

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Project description:BackgroundSupraventricular tachycardia is a group of rhythm disturbances that affect 1 in 300-1200 Australian children annually. The differentiation of supraventricular tachycardia (SVT) symptoms and age of onset according to their subtype is not well understood in the pediatric population. Most studies rely on ECG criteria only to characterize the subtype of the SVT, which is not applicable to all subtypes. The purpose of this study was to identify the symptoms and ages of onset of SVT subtypes, and to analyze whether ethnicity or severity correlated with the SVT subtype confirmed in an invasive Electrophysiology (EP) study.MethodsA retrospective analysis and prospective survey evaluated 364 patients who underwent an EP study at The Royal Children's Hospital, Melbourne between 2009 and 2015. Age of onset, symptoms, and ethnicity were collected by phone survey or medical records in addition to EP study diagnostic data, medication status, and follow-up information about their symptom status following EP procedure. Patients were grouped according to their SVT subtype. Data analysis was performed using chi-squared, Fisher's exact, and ANOVA statistical tests to determine associations between SVT substrates.ResultsTwo hundred and thirty-three suitable cases of SVT were identified (131 men, 102 women) aged between 0 and 18 years. Atrioventricular Reentrant Tachycardia (AVRT) (n = 153) was the most common SVT subtype, followed by Atrioventricular Nodal Reentrant Tachycardia (AVNRT) (n = 55), Atrial Tachycardia (AT) (n = 17), and other SVT subtypes (n = 8) which included Atrial Fibrillation, Atrial Flutter, and Junctional Tachycardia. There was a male predominance in all subtypes, except for AVNRT. AVNRT patients had palpitations, dyspnoea, dizziness, and anxiety more than any other group, AVRT patients complained of vomiting most and patients with AT had the most fatigue. The mean age of symptom onset varied among groups, being earlier in AVRT, later in AVNRT with a significant difference between AVRT with unidirectional retrograde accessory pathway (URAP) and AVNRT subtypes (P < 0.01).ConclusionSome specific symptoms were strong discriminators between different SVT subtypes. Ethnicity did not have strong correlations with SVT subtype incidence. This study was able to show clinical differences among children with SVT due to AVRT (URAP) compared to AVNRT, allowing the prognosis and intended management of pediatric SVT to be anticipated by less invasive means.

Project description:IntroductionBlack and Minoritised Ethnicity (BME) occupational therapists face lower career progression opportunities and mentoring is one possible intervention that may offer support. BME persons may have different expectations and experiences from their mentor, and research exploring their perceptions of mentoring is important. In Occupational Therapy there is a growing awareness of the need to be true to the values of social justice. The aim of this research is to learn about BME occupational therapists' experiences and perceptions of mentoring for career progression.MethodsFour online focus groups involving 19 participants from the UK were held, discussions were facilitated by researchers using a topic guide. Participants responses were analysed, and codes were brought together to create Themes including career progression and role models, mentoring relationship, similarity with the mentor and outcomes from mentoring.ResultsThe study highlighted that trust is integral to effective mentoring relationships and BME occupational therapists want mentors who they can trust with their experiences and stories. Compatibility between mentors and mentees in terms of personality, values, and working styles is crucial for effective mentoring relationships. Providing opportunities for networking, acknowledging good work and giving permission were all seen as outcomes from good mentoring. The absence of BME role models and ingroup bias were also reported as issues to be addressed.ConclusionThis study explores the perception of mentorship as a mechanism for career advancement in occupational therapists from BME backgrounds, with these results transferable to other health and care professional groups. We recommend the creation of a mentoring charter for BME healthcare workers in the United Kingdom to ensure that those from BME backgrounds feel supported, mentored, and provided with equitable access to resources, including adequate mentoring and networking opportunities.

Dataset Information

Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

Publications

Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

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